Non-invasive enzyme screen for tissue remodelling associated conditions

ABSTRACT

Methods and kits for diagnosing the presence of and prognosing the appearance of tissue remodelling-associated conditions, involving the presence of enzyme complexes in a biological sample, are disclosed. In particular, the method pertains to diagnosing the presence of or prognosing appearance of metastatic cancer by the identification of high molecular weight enzyme complexes comprising MMPs.

RELATED APPLICATIONS

The present application claims priority to U.S. provisional applicationSer. No. 60/240,489 filed on Oct. 13, 2000, the contents of which areexpressly incorporated herein by reference. This application is alsorelated to Ser. No. 08/639,373 filed on Apr. 26, 1996, (abandoned), U.S.Pat. No. 6,037,138, and Ser. No. 09/469,637 (pending), the entirecontents of each are expressly incorporated herein by reference. Thecontents of Yan, L. et al. (2001) J. Biol. Chem. 276: 37258-37265 areexpressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

Matrix metalloproteinases (MMP) are a family of endopeptidases whoseactivities depend on metal ions, such as Zn⁺⁺ and Ca⁺⁺. Collectively,MMPs are capable of degrading all the molecular components ofextracellular matrix (ECM), the barrier separating the tumor cells fromthe normal surrounding tissues, which is disassembled as part of themetastatic process (Lochter, A., et al. (1998) Ann N Y Acad. Sci. 857:180-93). MMPs have been shown to play important roles in a variety ofbiological as well as pathological processes, especially in tumor cellinvasion and metastasis (Kleiner, D. E. and Stetler-Stevenson, W. G.(1999) Cancer Chemother Pharmacol. 43: S42-51). Overproduction of MMPsby tumor cells or surrounding stromal cells has been correlated with themetastatic phenotype. In particular, U.S. Ser. No. 09/469,637, thecontents of which are herein incorporated by reference in theirentirety, teaches that intact and biologically active MMPs can bedetected in biological samples of cancer patients and are independentpredictors of disease status. The MMP activities detected in U.S. Ser.No. 09/469,637 include, for example, MMP-9 (92 kDa, gelatinase B, typeIV collagenase, EC3.4.24.35) and MMP-2 (72 kDa, gelatinase A, type IVcollagenase, EC3.4.24.24). Both of these MMPs have been shown to beindependent predictors of tissue remodeling-associated conditions, e.g.,cancer. In addition to these two major gelatinase species, several MMPactivities with molecular sizes of equal to, or greater than, 150 kDawere observed and were referred to as high molecular weight (hMW) MMPs.Elevated MMP levels in biological fluids, including serum, plasma, andurine from animals bearing experimental tumors or from cancer patientshave also been reported in several other studies (Nakajima, M., et al.,(1993) Cancer Res. 53: 5802-7; Zucker, S., et al.(1994) Ann N Y Acad.Sci. 732: 248-62; Baker, T., et al. (1994) Br J. Cancer. 70: 506-12;Garbisa, S., et al. (1992) Cancer Res. 52: 4548-9, 1992).

SUMMARY OF THE INVENTION

With the advances in cancer therapies, early diagnosis and/or prognosisare becoming increasingly important for the disease outcome.Accordingly, the present invention characterizes the molecular identityof hMW MMPs found in biological samples of subjects diagnosed withtissue remodelling-associated diseases, e.g., cancer, and provides earlydiagnosis/prognosis of such diseases. With the identification of thesehMW MMPS, e.g., high molecular weight enzyme complexes, the presentinvention facilitates the development of non-invasive diagnostic and/orprognostic methods to predict tissue remodelling-associated diseases,such as cancer.

The present invention provides methods and kits for detecting biologicalmarkers, e.g., high molecular weight enzyme complexes, to non-invasivelymonitor the diagnosis and prognosis of tissue remodelling-associatedconditions, e.g., cancers. Tissue remodelling-associated conditionsencompassed by such methods include diseases such as prostate cancer,breast cancer, ovarian cancer, brain tumors, arthritic conditions,obstructive conditions, and ulcerative conditions. The methods of theinstant invention use biological fluid samples, e.g., urine samples,that may be obtained by personnel without medical training, and do notrequire visiting a clinic or hospital. The statistical associationbetween positive results and occurrence of tissue remodelling-associatedconditions are applied to early diagnoses of the appearance of theseconditions, and to prognoses of changes in these conditions.

In one embodiment, the present invention provides non-invasive methodsfor facilitating the diagnosis of a subject for a tissueremodelling-associated condition. Such methods include obtaining abiological sample from a subject, and detecting a high molecular weightenzyme complex in the biological sample. The methods further includecorrelating the presence or absence of the high molecular weight enzymecomplex with the presence or absence of a tissue remodelling-associatedcondition, thereby facilitating the diagnosis of the subject for atissue remodelling-associated condition.

In another embodiment, the tissue remodelling-associated condition iscancer, e.g., organ-confined prostate cancer, metastatic prostatecancer, cancer found in cells of epithelial origin, mesodermal origin,endodermal origin or hematopoietic origin, and cancer selected from thegroup consisting of cancers of the nervous system, breast, retina, lung,skin, kidney, liver, pancreas, genito-urinary tract, andgastrointestinal tract. In another embodiment, the tissueremodelling-associated condition is an arthritic condition, anobstructive condition, or a degenerative condition.

In still another embodiment, the high molecular weight enzyme complexcomprises a protease, e.g., a serine protease, e.g., a matrixmetalloproteinase, e.g., an MMP-9.

In yet another embodiment, the high molecular weight enzyme complexfurther comprises a lipocalin, e.g., NGAL, and/or a TIMP, e.g., TIMP-1.

In still another embodiment, the high molecular weight enzyme complexcomprises an enzyme complexed with itself to form a multimer, e.g., adimer or a trimer. Such a multimer can further be complexed with alipocalin, e.g., NGAL, and/or a TIMP, e.g., TIMP-1.

In still yet another embodiment, the molecular weight of the highmolecular weight enzyme complex is at least about 115 kDa to at leastabout 125 kDa. In another embodiment, the molecular weight of the highmolecular weight enzyme complex is at least about 150 kDa.

In another embodiment, the methods of the present invention includeobtaining a biological sample from a subject and detecting lipocalin inthe biological sample. Such methods further include correlating thepresence or absence of the lipocalin with the presence or absence of atissue remodelling-associated condition, thereby facilitating thediagnosis of the subject for a tissue remodelling-associated condition.

In still another embodiment, the present invention provides kits forfacilitating the diagnosis and prognosis of a tissueremodelling-associated condition. Such kits include a container having areagent for detecting a high molecular weight enzyme complex in abiological sample and instructions for using the reagent for detectingthe high molecular weight enzyme complex which facilitates the diagnosisand prognosis of a tissue remodelling-associated condition.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a substrate gel electrophoresis and NGAL Western blotanalysis of urine samples. A. Substrate gel electrophoresis of MMPs inurine samples: 50 μl of untreated urine samples were analyzed for MMPactivities. Four major gelatinase activities were detected with apparentmolecular masses of approximately 200,000, 125,000, 92,000, and 72,000.Their identities are marked with arrows on right. The molecular sizemarkers are Perfect Protein Markers (Novagen, Madison, Wis.) with sizesof 150 kDa, 100 kDa, 75 kDa, and 50 kDa (arrows on left). B. 20 μl ofconcentrated urine samples were separated on a 4-15% SDS-PAGE gel undernon-reducing conditions. Western blot analysis was carried out using apolyclonal antibody against human NGAL. The molecular size markers areKaleidoscope Prestained Standards (Bio-Rad, Hercules, Calif.) with sizesof 126 kDa, 90 kDa, 44 kDa, 34 kDa, and 17 kDa (arrows on left).

FIG. 2 shows a Western blot and substrate gel electrophoresis of urinesamples and purified human neutrophil MMP-9/NGAL complex. A. NGALWestern blot analysis: A concentrated urine sample containing the 125kDa MMP activity, together with purified human neutrophil MMP-9/NGAL,were separated by 4-15% SDS-gel electrophoresis under non-reducingconditions, and subsequently subjected to Western blot analysis using apolyclonal antibody against human NGAL. The 125 kDa MMP-9/NGAL complexis marked (arrow on right). B. Substrate gel electrophoresis: The sameurine sample and purified human neutrophil MMP-9/NGAL complex wereanalyzed with substrate gel electrophoresis. The positions of MMP-9dimer (200 kDa), MMP-9/NGAL (125 kDa), MMP-9 (92 kDa), and MMP-2 (72kDa) are denoted with arrows on right. The molecular size markers arePerfect Protein Markers (Novagen, Madison, Wis.) with sizes of 150 kDa,100 kDa, 75 kDa, and 50 kDa (arrows on left).

FIG. 3 shows an immunoprecipitation of the 125 kDa MMP activity usinganti-NGAL antibody. 50 μl of urine samples (1:1 v/v diluted with RIPA)containing the 125 kDa MMP activity were mixed with 1.0, 0.1 or 0.01 μlof anti-NGAL antibody or a control antibody. After incubating on ice forthirty minutes, the antibody-antigen complexes were removed usingZysorbin. The supernatants were subjected to substrate gelelectrophoresis to detect the remaining MMP activities. The increasedMMP-2 activity observed in the sample treated with 1.0 μl of the controlantibody was the endogenous MMP-2 activity from the serum.

FIG. 4 shows reconstitution of MMP-9/NGAL complexes in vitro. A.Recombinant human MMP-9 and NGAL were diluted in gelatinase buffers withdifferent pH values and were subsequently mixed in a molar ratio of 1:10(proMMP-9 to NGAL). In vitro reconstitution was carried out at 37° C.for one hour. 10 μM proMMP-9 was loaded in each lane. Purified humanneutrophil MMP-9/NGAL was included as a control. B. Recombinant humanMMP-9 and NGAL were diluted in normal urine containing no MMP activitiesand were subsequently mixed in different molar ratios (proMMP-9 toNGAL=2:1, 1:5, 1:10, 1:20). After one hour incubation at 37° C.,MMP-9/NGAL complex formation was analyzed using substrate gelelectrophoresis. The positions of the 125 kDa and 115 kDa MMP-9/NGALactivity are respectively denoted with the arrow and the arrowhead onright. The molecular size markers are Perfect Protein Markers (Novagen,Madison, Wis.) with sizes of 150 kDa, 100 kDa, 75 kDa, and 50 kDa(arrows on left).

DETAILED DESCRIPTION OF THE INVENTION

The present invention features non-invasive methods for facilitating thediagnosis of a subject for a tissue remodelling-associated condition(TRAC), especially cancers, obstructive and degenerative conditions, andarthritic conditions. Detection of a pattern of enzyme complexes, e.g.,high molecular weight (hMW) enzyme complexes, in a biological samplefrom a subject is used to facilitate diagnosis and prognosis of a TRAC.

The language “high molecular weight enzyme complex” includes an enzymeassociated with or bound to another molecule wherein the complex has ahigh molecular weight allowing it to be used for its intended functionof the present invention. Examples of enzyme complexes include, amongothers, an enzyme bound to another enzyme, an enzyme bound to an enzymeinhibitor, and an enzyme bound to a protein binding molecule, e.g., alipocalin. Enzyme complexes which comprise enzymes bound to themselves,e.g., multimers, e.g., dimers and trimers, are also encompassed by thepresent invention.

High molecular weight enzyme complexes include enzyme complexes whichhave a molecular weight of at least about 115 kDa, e.g., at least about120 kDa, e.g., at least about 125 kDa, e.g., at least about 130 kDa,e.g., at least about 135 kDa, and, e.g., at least about 140 kDa. Highmolecular weight enzyme complexes which have a molecular weight of atleast about 145 kDa, e.g., at least about 150 kDa, and greater than 150kDa are also included.

The ranges of high molecular weight values intermediate to those listedalso are intended to be part of this invention, e.g. at least about 115kDa to at least about 120 kDa, at least about 120 kDa to at least about125 kDa, at least about 125 kDa to at least about 130 kDa, at leastabout 130 kDa to at least about 135 kDa, at least about 135 kDa to atleast about 140 kDa, at least about 140 kDa to at least about 145 kDa,and at least about 145 kDa to at least about 150 kDa. For example,ranges of high molecular weight values using a combination of any of theabove values recited as upper and/or lower limits are intended to beincluded.

In one embodiment of the invention, the high molecular weight enzymecomplex does not have a molecular weight of 115 kDa. In anotherembodiment, the high molecular weight enzyme complex does not includeNGAL. In another embodiment, the high molecular weight enzyme complexdoes not include a progelatinase B enzyme. In yet another embodiment ofthe invention, the high molecular weight enzyme complex does not includea progelatinase B enzyme associated with NGAL.

The term “enzyme” is art recognized and includes protein catalysts ofchemical reactions. Enzymes can be a whole intact enzyme or portions orfragments thereof. The enzymes encompassed by the enzyme complexes ofthe current invention include naturally occurring enzymes thatcatalytically degrade proteins, i.e. the enzymes known as proteases orproteinases. By proteinase is meant a progressive exopeptidase thatdigest proteins by removing amino acid residues from either the Nterminal or C terminal which reaction proceeds to achieve significantdegradation, or an endopeptidase which destroys the amide bond betweenamino acid residues with varying degrees of residue specificity. Theterm “protease” may also include the highly specific amino acidpeptidases that remove a single amino acid from an N terminus or Cterminus of a protein. Examples are alanine aminopeptidase (EC 3.4.11.2)and leucine aminopeptidase (EC 3.4.11.1), which remove alanine orleucine, respectively, from the amino terminus of a protein that mayhave alanine and leucine, respectfully, at the amino terminus. Themolecular weights of the enzymes comprising the enzyme complexes of theinvention include, but not limited to, molecular weights in the range ofapproximately 72 kDa, approximately 92 kDa, approximately 115 kDa toapproximately 125 kDa and approximately 150 kDa or greater. The term“enzyme” includes polymorphic variants that are silent mutationsnaturally found within the human population.

In one embodiment, the enzyme complexes of the present inventioncomprise proteases or proteinases. The term proteases (and itsequivalent term proteinases) is intended to include those endopeptidasesand progressive exopeptidases that are capable of substantially reducingthe molecular weight of the substrate and destroying its biologicalfunction, especially if that biological function of the substrate is tobe a structural component of a matrix barrier. Amino acid peptidasessuch as alanine aminopeptidase and leucine aminopeptidase are alsobroadly included among proteases, however do not share the property ofsignificantly reducing the molecular weight of the substrate protein.

Many thousands of proteases occur naturally, and each may appear atdifferent times of development and in different locations in anorganism. The invention herein features enzymes of the class of thematrix metalloproteinases (MMPs, class EC 3.4.24). These enzymes, whichrequire a divalent cation for activity, are normally expressed early inthe development of the embryo, for example, during hatching of an zygotefrom the zona pellucida, and again during the process of attachment ofthe developing embryo to the inside of the uterine wall. Enzymeactivities such as N-acetylglucosaminidase (EC 3.2.1.50) appear in urinein the case of renal tubular damage, for example, due to diabetes (Carr,M. (1994) J. Urol. 151(2):442-445; Jones, A., et. al. (1995) Annals.Clin. Biochem., 32:68-62). That these activities appear in urine as aresult of renal tubular damage is irrelevant to the present invention asdescribed herein.

The term “matrix-digesting enzyme” includes an enzyme capable ofdigesting or degrading a matrix, e.g., a mixture of proteins andproteoglycans that comprise a layer in a tissue on which certain typesof cells are found. Matrix-digesting enzymes are expressed during stagesof normal embryogenesis, pregnancy and other processes involving tissueremodelling. In addition, some of these enzymes, for example some matrixmetalloproteinases (MMPs), degrade the large extracellular matrixproteins of the parenchymal and vascular basement membranes that serveas mechanical barriers to tumor cell migration. These MMPs are producedin certain cancers and are associated with metastasis (Liotta, L. A., etal. (1991) Cell 64:327-336). Examples of MMPs are the type IVcollagenases, e.g., MMP-2 (gelatinase A. EC 3.4.24.24) and MMP-9(gelatinase B, 3.4.24.35), and stromelysins (EC 3.4.24.17 and3.4.24.22). Some MMPs are specifically inhibited by molecules calledtissue inhibitors of metalloproteinases (TIMPs, Woessner, J. F., Jr.(1995) Ann. New York Acad. Sci., 732:11-21), which also may beoverproduced by tumor cells, however under certain conditions enzymeactivity is in molar excess over the TIMPs (Freeman, M. R. et al. (1993)J. Urol. 149:659; Lu, X. et al. (1991) Cancer Res. 51:6231-6235;Kossakowska, A. E. et al. (1991) Blood 77:2475-2481). Accordingly, inone embodiment of this invention, the enzyme complexes of the presentmethods comprise an inhibitor of the enzyme (TIMPS, e.g., TIMP-1 orTIMP-2). The detection of an inhibitor can be accomplished usingart-recognized techniques. Many of MMPs are translated as pro-enzymes,and may be found in a variety of structures, with ranges of molecularweights including smaller forms (45 kDa, 55 kDa, 62 kDa), and largerforms (72 kDa, 82 kDa, 92 kDa, and higher polymers such as 150 kDa andgreater).

In another embodiment of the invention, the high molecular weight enzymecomplex comprises a protein binding molecule, e.g., a lipocalin.Lipocalins are small secreted proteins that bind small, hydrophobicmolecules to form molecular complexes. Lipocalins are implicated in avariety of functions including, among others, regulation of the immuneresponse, e.g., lipocalins can exert certain immunomodulatory effects invitro. It has been shown that neutrophil lipocalin covalently attachesto human neutrophil gelatinase (type IV collagenase) thus formingNeutrophil Gelatinase-Associated Lipocalin (NGAL) (Treibel et al. (1992)and Kjelsen et al. (1993)) although most of the protein is secreted inuncomplexed form. These authors prose a regulatory role for NGAL on theaction of the gelatinase.

In another embodiment, the present invention includes methods ofdetecting a lipocalin, e.g., NGAL, as an indicator of a TRAC. Suchlipocalins can be detected in a biological sample as an isolatedlipocalin or as multimers of lipocalins, e.g., dimers and trimers.

The tissue remodelling conditions that can be monitored by the methodsof this invention include a variety of types of cancer; moreover, theenzymes are suitable for diagnosis of other tissue remodellingconditions, such as arthritis, degenerative conditions, and obstructiveconditions. The invention provides non-invasive methods for diagnosingthese conditions by assay for enzyme complexes, e.g., hMW enzymecomplexes, in biological fluids.

The methods of this invention embody detection of enzymes in urine, fordiagnosis and prognosis of cancer. The invention also relates todiagnosis and prognosis of metastatic prostate cancer. The varieties ofcancer suitable for diagnosis by the methods of this invention include,among others, cancers of epithelial origin, for example, cancers of thenervous system, breast, retina, lung, skin, kidney, liver, pancreas,genito-urinary tract, ovarian, uterine and vaginal cancers, andgastrointestinal tract cancers, which form in cells of epithelialorigin. Using the methods described here, cancers of mesodermal andendodermal origin, for example, cancers arising in bone or inhematopoietic cells, are also diagnosed.

The term “subject,” as used herein, includes a living animal or human inneed of diagnosis or prognosis for, or susceptible to, a condition, inparticular an “tissue remodelling-associated condition” as definedbelow. The subject is an organism capable of responding to tissueremodelling signals such as growth factors, under some circumstances,the subject is susceptible to cancer and to arthritis. In oneembodiment, the subject is a mammal, including humans and non-humanmammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, andmice. In one embodiment, the subject is a human. The term “subject” doesnot preclude individuals that are entirely normal with respect to tissueremodelling-associated conditions or normal in all respects. The subjectmay formerly have been treated surgically or by chemotherapy, and may beunder treatment by hormone therapy or have been treated by hormonetherapy in the past.

The term “patient,” as used herein, includes a human subject who haspresented at a clinical setting with a particular symptom or symptomssuggesting one or more diagnoses. A patient may be in need of furthercategorization by clinical procedures well-known to medicalpractitioners of the art (or may have no further disease indications andappear to be in any or all respects normal). A patient's diagnosis mayalter during the course of disease progression, such as development offurther disease symptoms, or remission of the disease, eitherspontaneously or during the course of a therapeutic regimen ortreatment. Thus, the term “diagnosis” does not preclude differentearlier or later diagnoses for any particular patient or subject. Theterm “prognosis” includes an assessment for a subject or patient of aprobability of developing a condition associated with or otherwiseindicated by presence of one or more enzymes in a biological sample,e.g., in urine.

The term “biological sample” includes biological samples obtained from asubject. Examples of such samples include urine, blood taken from aprick of the finger or other source such as intravenous, blood fractionssuch as serum and plasma, feces and fecal material and extracts, saliva,cerebrospinal fluid, amniotic fluid, mucus, and cell and tissue materialsuch as cheek smear, Pap smear, fine needle aspiration, sternumpuncture, and any other biopsied material taken during standard medicaland open surgical procedures.

The term “invasiveness” as used here with respect to metastatic cancer(Darnell, J. (1990) Molecular Cell Biology, Third Ed, W.H. Freeman, NY)is distinct from the use of the term “invasive” to describe a medicalprocedure, and the distinction is made in context. “Invasive” for amedical procedure pertains to the extent to which a particular procedureinterrupts the integrity of the body. “Invasiveness” ranges from fullynon-invasive, such as collection of urine or saliva; to mildly invasive,for example a Pap smear, a cheek scrape or blood test, which requirestrained personnel in a clinical setting; to more invasive, such as asternum marrow collection or spinal tap; to extensively invasive, suchas open surgery to detect the size and nature of tumors by biopsy ofmaterial, taken for example during brain surgery, lung surgery, ortransurethral resection in the case of prostate cancer.

Cancer or neoplasia is characterized by deregulated cell growth anddivision. A tumor arising in a tissue originating from endoderm orexoderm is called a carcinoma, and one arising in tissue originatingfrom mesoderm is known as a sarcoma (Darnell, J. (1-990) Molecular CellBiology, Third Ed., W.H. Freeman, NY). A current model of the mechanismfor the origin of a tumor is by mutation in a gene known as an oncogene,or by inactivation of a second tumor-suppressing genes (Weinberg, R. A.(September 1988) Scientific Amer. 44-51). The oncogenes identified thusfar have arisen only in somatic cells, and thus have been incapable oftransmitting their effects to the germ line of the host animal. Incontrast, mutations in tumor-suppressing genes can be identified in germline cells, and are thus transmissible to an animal's progeny. Examplesof cancers include cancers of the nervous system, breast, retina, lung,skin, kidney, liver, pancreas, genito-urinary tract, gastrointestinaltract, cancers of bone, and cancers of hematopoietic origin such asleukemias and lymphomas. In one embodiment of the present invention, thecancer is not a cancer of the bladder.

An arthritic condition such as rheumatoid arthritis is an example of aTRAC since the disease when chronic is characterized by disruption ofcollagenous structures (J. Orten et al. (1982) Human Biochemistry, TenthEd., C.V. Mosby, St. Louis, Mo.). Excess collagenase is produced bycells of the proliferating synovium. Other TRAC conditions such asulcerative, obstructive and degenerative diseases are similarlycharacterized by alterations in the enzymes of metabolism of structuralproteins.

The term “electrophoresis” is used to indicate any separation system ofmolecules in an electric field, generally using an inert support systemsuch as paper, starch gel, or polyacrylamide. The electrophoresismethods with polyacrylamide gels and the sodium dodecyl sulfatedenaturing detergent are described in the Examples below. The protocolsare not intended to exclude equivalent procedures known to the skilledartisan. Other SDS polyacrylamide procedures, known to the skilledartisan, may be used, e.g., a single polyacrylamide concentration suchas 10%, may be substituted for the gradient in the separation gel. Thephysical support for the electrophoretic matrix may be capillary tubesrather than glass plates. Details of several SDS-polyacrylamide gelelectrophoresis systems are described in many review articles andbiotechnology manuals (e.g., Maniatis, T., Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). The method is not limited to use of SDS and otherdetergents. Further, electrophoresis in the absence of detergents may beemployed. Proteins may be separated under non-denaturing conditions, forexample in the presence of urea on a polyacrylamide matrix (Maniatis,supra), or by charge, for example by the procedure of iso-electricfocussing.

In using an electrophoretic technique for separation of enzymes, theelectrophoretogram may be developed as a zymogram. The term “zymography”is meant here to include any separations system utilizing a chemicallyinert separating or support matrix, that allows detection of an enzymefollowing electrophoresis, by exposing the matrix of the separationssystem to conditions that allow enzyme activity and subsequentdetection. More narrowly, the term zymography designates incorporationof an appropriate substrate for the enzyme of interest into the inertmatrix, such that exposing the matrix to the conditions of activityafter the electrophoresis stop yields a system to visualize the preciselocation, and hence the mobility, of the active enzyme. By techniqueswell-known to the skilled artisan, the molecular weights of proteins arecalculated based on mobilities derived from positions on a zymogram.Such techniques include comparison with molecular weight standards, themobilities of which are determined from general protein stains or frompre-stains specific to those standards, and comparison with positivecontrols of purified isolated enzymes of interest, which are visualizedby the technique of the zymogram, i.e., enzyme activity.

In particular, substrates for detection of proteases by zymography areincluded in the electrophoresis matrix. For type IV collagenases, thenatural substrate is a type IV collagen and gelatin, a type I collagenderivative, is used for the zymography substrate in the Examplespresented herein. However other proteins that are suitable for detectionof further proteases of interest in TRAC diagnosis, for example, includefibronectin; vitronectin; collagens of types I through III and V throughXII; procollagens; elastin; laminin; plasmin; plasminogen; entactin;nidogen; syndecan; tenascin; and sulfated proteoglycans substituted withsuch saccharides as hyaluronic acid, chondroitin-6-sulfate,condroitin-4-sulfate, heparan sulfate, keratan sulfate, and dermatansulfate and heparin. Further, convenient inexpensive substrate proteinssuch as casein, which may not be the natural target of a protease ofinterest, but are technically appropriate, are included as suitablesubstrate components of the zymography techniques of the presentinvention. Chemically synthesized mimetics of naturally occurringprotein substrates are also potential zymography substrates, and mayeven be designed to have favorable properties, such chromogenic orfluorogenic ability to produce a color or fluorescent change uponenzymatic cleavage.

Zymography may be adapted to detection of a protease inhibitor in thebiological sample. Since a variety of natural MMP inhibitors areelaborated, such as TIMP-1 and TIMP-2, and are found to be deregulatedduring TRAC situations, the present invention includes detection ofenzyme complexes which comprise enzyme inhibitors, e.g., TIMPs. Thus forexample, a “reporter enzyme” for which an enzyme inhibitory activity isbeing measured, may be incubated with each biological sample obtained bysubjects and patients, in one or more quantities corresponding to one ormore aliquots of sample, prior to electrophoresis. This enzyme isomitted from one aliquot of the biological sample. The inhibitorypresence in the sample is detected as disappearance or decrease of thereporter enzyme band from the developed zymogram. Alternatively,functional enzyme activity assays which include in the reaction mix aknown level of active enzyme, to which is added aliquots of experimentalsamples with putative inhibitory activity, can detect the presence ofinhibitors.

Further, the enzymes of tissue remodelling extend to enzyme activitiesbeyond those of proteolytic activity. For example, enzymes that aresubstituted with residues such as glycosyl, phosphate, sulfate, lipidsand nucleotide residues (e.g. adenyl) are well-known to those skilled inthe art. These residues are in turn added or removed by other enzymes,e.g., glycosidases, kinases, phosphatases, adenyl transferases, etc.Convenient detection methods for the presence of such activities forTRAC diagnosis and prognosis are readily developed by those with skillin the art, and are intended to comprise part of the invention here.

The zymogram as described in the Examples herein is developed by use ofa general stain for protein, in this case, Coomassie Blue dye. Thedevelopment is possible with general protein stains, e.g., Amido Blackdye, and SYPRO Orange stain (Biorad Laboratories, Hercules, Calif.94537). Further, enzyme activity may be detected by additionaltechniques beyond that of a clear zone of digestion in a stained matrix,for example, by absence of areas of radioactivity with a radio-labelledsubstrate, by change in mobility of a radio-labelled substrate, or byabsence of or change in mobility of bands of fluorescence or colordevelopment with use of fluorogenic or chromogenic substrates,respectfully.

Quantitative densitometry can be performed with zymograms by placing thegel directly on an activated plate of a Molecular Dynamicsphosphorimager (Molecular Dynamics, 928 East Arques Ave., Sunnyvale,Calif. 94086), or with a Datacopy G8 plate scanner attached to aMacIntosh computer equipped with an 8-bit videocard and McImage (XeroxImaging Systems). Background measurements, areas of the gel separatefrom sample lanes, can similarly be scanned, and values subtracted fromthe readings for enzyme activities.

Another electrophoretically-based technique for analysis of a biologicalsample for presence of specific proteins is an affinity-based mobilityalteration system (Lander, A. (1991) Proc. Natl. Acad. Sci. U.S.,88(7):2768-2772). An MMP or other type of enzyme of interest might bedetected, for example, by inclusion of a substrate analog that bindsessentially irreversibly to the enzyme, hence decreasing the mobility.The affinity material is present during electrophoresis, and isincorporated into the matrix, so that detection of the enzyme ofinterest occurs as a result of alteration of mobility in contrast tomobility in the absence of the material. Yet another technique ofelectrophoretic protein separation is based on the innate charge of aprotein as a function of the pH of the buffer, so that for any proteinspecies, there exists a pH at which that protein will not migrate in anelectric field, or the isoelectric point, designated pI. Proteins of abiological sample, such as a urine sample, may be separated byisoelectric focussing, then developed by assaying for enzymatic activityfor example by transfer to material with substrate, i.e., zymography.Electrophoresis is often used as the basis of immunological detections,in which the separation step is followed by physical or electrophoretictransfer of proteins to an inert support such as paper or nylon (knownas a “blot”), and the blotted pattern of proteins may be detected by useof a specific primary binding (Western blot) by an antibody followed bydevelopment of bound antibodies by secondary antibodies bound to adetecting enzyme such as horse radish peroxidase. Additionalimmunological detection systems for TRAC enzyme complexes are nowdescribed in detail below.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the componentsin the methods and kits of the invention. Antibodies can be fragmentedusing conventional techniques and the fragments screened for utility inthe same manner as described above for whole antibodies. For example,F(ab)₂ fragments can be generated by treating an antibody with pepsin.The resulting F(ab)₂ fragment can be treated to reduce disulfide bridgesto produce Fab fragments. The term “antibody” is further intended toinclude single chain, bispecific and chimeric molecules. The term“antibody” includes possible use both of monoclonal and polyclonalantibodies (Ab) directed against a target, according to the requirementsof the application.

Polyclonal antibodies can be obtained by immunizing animals, for examplerabbits or goats, with a purified form of the antigen of interest, or afragment of the antigen containing at least one antigenic site.Conditions for obtaining optimal immunization of the animal, such as useof a particular immunization schedule, and using adjuvants e.g. Freund'sadjuvant, or immunogenic substituents covalently attached to theantigen, e.g. keyhole limpet hemocyanin, to enhance the yield ofantibody titers in serum, are well-known to those in the art. Monoclonalantibodies are prepared by procedures well-known to the skilled artisan,involving obtaining clones of antibody-producing lymphocyte, i.e. celllines derived from single cell line isolates, from an animal, e.g. amouse, immunized with an antigen or antigen fragment containing aminimal number of antigenic determinants, and fusing said clone with amyeloma cell line to produce an immortalized high-yielding cell line.Many monoclonal and polyclonal antibody preparations are commerciallyavailable, and commercial service companies that offer expertise inpurifying antigens, immunizing animals, maintaining and bleeding theanimals, purifying sera and IgG fractions, or for selecting and fusingmonoclonal antibody producing cell lines, are available.

Specific high affinity binding proteins, that can be used in place ofantibodies, can be made according to methods known to those in the art.For example, proteins that bind specific DNA sequences may be engineered(Ladner, R. C., et. al., U.S. Pat. No. 5,096,815), and proteins thatbind a variety of other targets, especially protein targets (Ladner, R.C., et. al., U.S. Pat. No. 5,233,409; Ladner, R. C., et al., U.S. Pat.No. 5,403,484) may be engineered and used in the present invention forcovalent linkage to a chelator molecule, so that a complex with aradionuclide may be formed under mild conditions. Antibodies and bindingproteins can be incorporated into large scale diagnostic or assayprotocols that require immobilizing the compositions of the presentinvention onto surfaces, for example in multi-well plate assays, or onbeads for column purifications.

General techniques to be used in performing various immunoassays areknown to those of ordinary skill in the art. Moreover, a generaldescription of these procedures is provided in U.S. Pat. No. 5,051,361which is incorporated herein by reference, and by procedures known tothe skilled artisan, and described in manuals of the art (Ishikawa, E.,et. al. (1988) Enzyme Immunoassay Igaku-shoin, Tokyo, NY; Hallow, E. andD. Lane, Antibodies: A Laboratory Manual, CSH Press, NY). Examples ifseveral immunoassays are given discussed here.

Radioimmunoassays (RIA) utilizing radioactively labeled ligands, forexample, antigen directly labeled with ³H, or ¹⁴C, or ¹²⁵I, measurepresence of MMP's as antigenic material. A fixed quantity of labeled MMPantigen competes with unlabeled antigen from the sample for a limitednumber of antibody binding sites. After the bound complex of labeledantigen-antibody is separated from the unbound (free) antigen, theradioactivity in the bound fraction, or free fraction, or both, isdetermined in an appropriate radiation counter. The concentration ofbound labeled antigen is inversely proportional to the concentration ofunlabeled antigen present in the sample. The antibody to MMP can be insolution, and separation of free and bound antigen MMP can beaccomplished using agents such as charcoal, or a second antibodyspecific for the animal species whose immunoglobulin contains theantibody to MMP. Alternatively, antibody to MMP can be attached to thesurface of an insoluble material, which in this case, separation ofbound and free MMP is performed by appropriate washing.

Immunoradiometric assays (IRMA) are immunoassays in which the antibodyreagent is radioactively labeled. An IRMA requires the production of amultivalent MMP conjugate, by techniques such as conjugation to aprotein e.g., rabbit serum albumin (RSA). The multivalent MMP conjugatemust have at least 2 MMP residues per molecule and the MMP residues mustbe of sufficient distance apart to allow binding by at least twoantibodies to the MMP. For example, in an IRMA the multivalent MMPconjugate can be attached to a solid surface such as a plastic sphere.Unlabeled “sample” MMP and antibody to MMP which is radioactivelylabeled are added to a test tube containing the multivalent MMPconjugate coated sphere. The MMP in the sample competes with themultivalent MMP conjugate for MMP antibody binding sites. After anappropriate incubation period, the unbound reactants are removed bywashing and the amount of radioactivity on the solid phase isdetermined. The amount of bound radioactive antibody is inverselyproportional to the concentration of MMP in the sample.

Other immunoassay techniques use enzyme labels such as horseradishperoxidase, alkaline phosphatase, luciferase, urease, andβ-galactosidase. For example, MMP's conjugated to horseradish peroxidasecompete with free sample MMP's for a limited number of antibodycombining sites present on antibodies to MMP attached to a solid surfacesuch as a microtiter plate. The MMP antibodies may be attached to themicrotiter plate directly, or indirectly, by first coating themicrotiter plate with multivalent MMP conjugates (coating antigens)prepared for example by conjugating MMP with serum proteins such asrabbit serum albumin (RSA). After separation of the bound labeled MMPfrom the unbound labeled MMP, the enzyme activity in the bound fractionis determined colorimetrically, for example by a multi-well microtiterplate reader, at a fixed period of time after the addition ofhorseradish peroxidase chromogenic substrate.

Alternatively, the antibody, attached to a surface such as a microtiterplate or polystyrene bead, is incubated with an aliquot of thebiological sample. MMP present in the fluid will be bound by theantibody in a manner dependent upon the concentration of MMP and theassociation constant between the two. After washing, the antibody/MMPcomplex is incubated with a second antibody specific for a differentepitope on MMP distal enough from the MMP-specific antibody binding sitesuch that stearic hindrance in binding of two antibodies simultaneouslyto MMP may be accomplished. For example, the second antibody may bespecific for a portion of the proenzyme sequence. The second antibodycan be labeled in a manner suitable for detection, such as byradioisotope, a fluorescent compound or a covalently linked enzyme. Theamount of labeled secondary antibody bound after washing away unboundsecondary antibody is proportional to the amount of MMP present in thebiological sample.

The above examples of immunoassays describe the use of radioactively andenzymatically labeled tracers. Assays also may include use offluorescent materials such as fluorescein and analogs thereof,5-dimethylaminonaphthalene-1-sulfonyl derivatives, rhodamine and analogsthereof, coumarin analogs, and phycobiliproteins such as allophycocyaninand R-phycoerythrin; phosphorescent materials such as erythrosin andeuropium; luminescent materials such as luminol and luciferin; and solssuch as gold and organic dyes. In one embodiment of the presentinvention, the biological sample is treated to remove low molecularweight contaminants.

In one embodiment of the present invention, the biological sample istreated to remove low molecular weight contaminants, for example, bydialysis. By the term “dialysis” this invention includes any techniqueof separating the enzymes in the sample from low molecular weightcontaminants. The Examples use Spectra/Por membrane dialysis tubing witha molecular weight cut-off (MWCO) of 3,500, however other products withdifferent MWCO levels are functionally equivalent. Other productsinclude hollow fiber concentration systems consisting of regeneratedcellulose fibers (with MWCO of 6,000 or 9,000) for larger volumes; amultiple dialyzer apparatus with a sample size for one to 5 ml; andmultiple microdialyzer apparatus, convenient for samples in plates with96 wells and MWCOs at 5,000, 8,000 and 10,000, for example. Theseapparatuses are available from PGC Scientific, Gaithersburg, Md., 20898.Those with skill in the art will appreciate the utility of multipledialysis units, and especially suitable for kits for reference lab andclinic usage. Other equivalent techniques include passage through acolumn holding a resin or mixture of resins suitable to removal of lowmolecular weight materials. Resins such as BioGel (BioRad, Hercules,Calif.) and Sepharose (Pharmacia, Piscataway, N.J.) and others arewell-known to the skilled artisan. The technique of dialysis, orequivalent techniques with the same function, are intended to remove lowmolecular weight contaminants from the biological fluids. While not anessential component of the present invention, the step of removal ofsuch contaminants facilitates detection of the disorder-associatedenzymes in the biological samples.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES

The following materials and methods were used throughout these Examples,set forth below.

Materials and Methods

Urine Sample Collection and Preparation—Urine sample collection wasperformed as described in Moses, M. A., et al. (1998) Cancer Res.58:1395-9, the contents of which are herein incorporated by reference intheir entirety. Samples were immediately frozen after collection andstored frozen at −20° C. until assay. Prior to analysis, specimenscontaining blood or leukocytes were excluded by testing for the presenceof blood and leukocytes using Multistix 9 Urinalysis Strips (Bayer,Elkhart, Ind.). The creatine concentrations of urine samples weredetermined using a commercial kit (Sigma Chemical Co., St. Louis, Mo.)according to manufacturer's instructions.

Substrate Gel Electrophoresis—Substrate gel electrophoresis wasperformed based on a previously described in U.S. Ser. No. 09/469,637with modifications. Original urine samples (50 μl) were mixed withnon-reducing sample buffer [4% sodium dodecyl sulfate (SDS), 0.15 M TrispH 6.8, 20% v/v glycerol, and 0.5% v/v bromphenol blue] and wereseparated on a 10% polyacrylamide gel containing 0.1% gelatin (Bio-Rad,Hercules, Calif.). After electrophoresis, gels were washed twice with2.5% Triton X-100 (15 minutes/each wash). Substrate digestion wascarried out by incubating the gel in 50 mM Tris-HCl (pH7.6) containing 5mM CaCl₂, 1 μM ZnCl₂, 1% Triton X-100, and 0.02% NaN₃ at 37° C. for 24hours. The gel was stained with 0.1% Coomassie Brilliant Blue R250(BioRad, Hercules, Calif.), and the location of gelatinolytic activitieswere detected as clear bands on the background of a uniform bluestaining.

Protein Electrophoresis and Western Blot Analysis—Urine samples wereconcentrated using an UltraFree-4 centrifugal filter device withmolecular weight cut off (MWCO) of 50 kDa (Millipore, Bedford, Mass.).Protein concentrations of the concentrated urine samples were determinedusing the MicroBCA method (Pierce, Rockford, Ill. 61105). Equal amountof proteins (20 μg) was loaded onto 4-15% gradient gels and separated bySDS-PAGE under non-reducing conditions. Resolved proteins wereelectrophoretically transferred to nitrocellulose membranes (TransBlot,Bio-Rad, Hercules, Calif.). The membranes were blocked with 5% low fatdry milk in TBS-T (.10 mM Tris, pH 7.2, 50 mM NaCl, 0.5% Tween 20) for 1hour at room temperature, followed by incubating with primary antibodyat 4° C. for 18 hours. Blots were washed 8 times with TBS-T (5minutes/wash) and incubated with 1:5000 dilution of horseradishperoxidase (HRP) conjugated secondary antibody (Vector Laboratories,Burlingame, Calif.) diluted in TBS-T containing 3% BSA for 1 hour atroom temperature. Labeled proteins were visualized with enhancedchemiluminescence (Amersham, Arlington Heights, Ill.). Purifiedpolyclonal antibodies against human NGAL were used at 1:100 dilution(Kjeldsen, L., et al. (1993)). Purified human neutrophil MMP-9/NGALcomplex was used as positive control (CalBiochem, La Jolla, Calif.).

Immunoprecipitation—Original urine samples containing the 125 kDa MMPactivity were mixed with equal volumes of RIPA buffer (150 mM NaCl, 1.0%NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 0.02%sodium azide). 50 μl diluted urine samples were mixed with increasingamount of the rabbit anti-human NGAL antibody or a control antibody.After incubating on ice for thirty minutes, samples were mixed with 5 μlRIPA-buffered Zysorbin (ZyoMed Laboratories, South San Francisco,Calif.). Followed an additional incubation on ice for thirty minutes,the antibody-antigen complexes were removed with a centrifugation at10,000 g for 5 minutes. The supernatants were subjected to substrate gelelectrophoresis to detect the remaining MMP activities.

In vitro reconstitution of MMP-NGAL complexes—Recombinant human proMMP-9(Oncogene, Cambridge, Mass.) was diluted with gelatinase buffer [50 mMSodium Acetate (pH=5.5) or 50 mM Tris-HCl (pH=7.0, 7.6, or 8.0)containing 5 mM CaCl₂, 1 μM ZnCl₂], to a final concentration of 10 μM.Recombinant human NGAL was purified as previously described and wasdiluted to 70 μM in the gelatinase buffer. ProMMP-9 was mixed with NGALin a molar ratio of 1:20 and was incubated at 37° C. for one hour. Theformation of MMP-9/NGAL complex was analyzed using substrate gelelectrophoresis. ProMMP-9 and NGAL were also individually diluted innormal control urine with no MMP activity. The possibility of MMP-9/NGALcomplex formation in urine was investigated by mixing proMMP-9 and NGALin moral ratios of 2:1, 1:5, 1:10 and 1:20. MMP-9/NGAL complex wasdetected using substrate gel electrophoresis.

Example 1

Substrate Gel Electrophoresis of MMP Activities in Urine Samples

MMP activities contained in urine samples were assayed using substrategel electrophoresis. 50 μl of freshly thawed urine sample was used foranalysis. At least four major MMP activities were readily detected inthese urine samples, with apparent molecular mass of 200,000, 125,000,92,000, and 72,000 (FIG. 1A). The 92 kDa and the 72 kDa MMP activitieshave previously been determined to be MMP-9 and MMP-2 respectively. The200 kDa MMP activities is in correspondence with the predicted molecularsize of MMP-9 dimer. The identity of the 125 kDa MMP is unclear. Whenanalyzed together with purified human MMP-9/NGAL complex fromneutrophil, the 125 kDa urinary MMP activity migrated in the sameposition as that of human neutrophil MMP-9/NGAL (FIG. 2A). This 125 kDaurinary MMP is an active complex of MMP-9 and NGAL. The identity ofthese gelatinolytic activities of being MMPs was confirmed by inhibitionstudies using 1,10-phenanthroline at a final concentration of 10 mM(data not shown).

Example 2

Western Blot Analysis of Urine Samples with Anti-NGAL Antibody

To further demonstrate the identity of the 125 kDa urinary MMP as acomplex of MMP-9 and NGAL, concentrated urine samples were subjected toWestern blot analysis using a purified antibody against human NGAL(Kjeldsen, L. (1993)). Under non-reducing conditions, a protein band of125 kDa was detected in urine samples containing the 125 kDa MMPactivity (FIG. 1B). Screening of urine samples from cancer patientsestablished a correlationship between the detection of MMP-9/NGALprotein complex and the presence-of the 125 kDa MMP activity (FIG. 1B).Using the purified anti-NGAL antibody, a 125 kDa protein band wasconsistently detected in urine samples containing the 125 kDa MMPactivity. The antibody also detected the presence of NGAL monomer (25kDa), dimer (50 kDa), and trimer forms (75 kDa) in all of the urinesamples analyzed. The specificity of the NGAL antibody was confirmedusing purified human neutrophil MMP-9/NGAL complex. Under non-reducingconditions, the antibody recognized the 125 kDa MMP-9/NGAL complex inthe concentrated urine sample, as well as the MMP-9/NGAL complexpurified from neutrophil (FIG. 2B). In addition to the MMP-9/NGALcomplex and the NGAL monomer, dimer and trimer complexes, several minorprotein bands with approximate molecular sizes of 150 kDa were alsodetected in the concentrated urine sample. Although their identities arecurrently unclear, they are most likely to be proteins thatnon-specifically cross-reacted with anti-NGAL antibody.

Example 3

Immunoprecipitation-Zymography

To further verify the identity of the 125 kDa MMP activity in urine,anti-NGAL antibody was used to immunoprecipitate any MMP activities thatexist in the complex form with NGAL in urine. As shown in FIG. 3,anti-NGAL antibody specifically immunoprecipitated the 125 kDa urinaryMMP activity, in a concentration-dependent manner. Increasing amounts ofthe 125 kDa urinary MMP activity was removed by the treatment withincreasing amounts of anti-NGAL antibody. When treated with 1.0 μl ofanti-NGAL antibody, the 125 kDa MMP activity was completely removed. Theanti-NGAL antibody had no effect on any other MMP activities, e.g., the200 kDa MMP-9 dimer, the 92 kDa MMP-9, or the 72 kDa MMP-2. Thespecificity of immunoprecipitation was also confirmed using a controlantibody which did not immunoprecipitate any of the MMP activities, evenat the highest concentration. The increase in MMP-2 activity in thesample treated with 1.0 μl of control antibody resulted from endogenousMMP-2 activity contained in the serum. Taken together these data supportour finding that the 125 kDa MMP activity in urine samples of cancerpatients is a complex of MMP-9 and NGAL.

Example 4

Re-Constitution of MMP-9/NGAL Complex In Vitro

The formation of MMP-9/NGAL complex was first investigated usinggelatinase buffer that contains cationic ions. Recombinant humanproMMP-9 and human NGAL were first diluted in gelatinase buffers withdifferent pH values (5.5, 7.0, 7.6 and 8.0). Diluted proMMP-9 and NGALwere subsequently mixed in a molar ratio of 1:10, to finalconcentrations of 2.6 μM and 26 μM respectively. After one hourincubation at 37° C., the formation of MMP-9/NGAL complexes wasmonitored using substrate gel electrophoresis. Mixing proMMP-9 and NGALgenerated a predominant MMP activity with a molecular size ofapproximately 115 kDa (FIG. 4A). Formation of the 115 kDa MMP-9/NGALcomplex occurred in buffers with pH values ranging from 5.5 to 8.0, thepH range of normal urine. However, the size of this predominant MMPactivity is not the same as that of purified human neutrophilMMP-9/NGAL. There is a minor MMP activity of 125 kDa, observed in pH7.0, 7.6 and 8.0 buffers. The possibility of MMP-9/NGAL complexformation in urine was directly studied by diluting proMMP-9 and NGAL innormal control urine. Diluted proMMP-9 and NGAL were mixed in differentmolar ratios (proMMP-9/NGLA=2:1, 1:5, 1:10 and 1:20) and incubated at37° C. for one hour. The formation of a 115 kDa MMP-9/NGAL complex wasreadily detected in all mixing ratios (FIG. 4B). No MMP activity wasdetected in the control urine used as a diluent.

Example 5

Modulation of MMP-9 Degradation by NGAL In Vitro

The effect of NGAL on MMP-9 degradation in vitro was studied by mixingMMP-9 (0.1μ) and NGAL (1/0μ) prior to incubation. MMP-9 degradation wasinhibited in the presence of NGAL resulting in a decrease in theenzymatic degradation rate as evidenced by an increase in the remainingamounts of enzyme at each time point compared with MMP-9 incubated byitself. Immunodepleted NGAL had no apparent protection of MMP-9. In thepresence of increasing amount of NGAL, degradation of MMP-9 decreasedand resulted in an increase in the remaining MMP-9 activity. NGALappears to be capable of protecting MMP-9 from degradation in adose-dependent manner, resulting in the preservation of MMP-9 activity.These data suggest a potential regulatory role for NGAL in modulatingMMP-9 activity, for example, NGAL may be involved in tumor progressionvia its interaction with MMP-9.

Example 6

Modulation of MMP-9 Degradation by NGAL In Cell Culture

The protective effect of NGAL on MMP-9 degradation was studied in cellculture using MDA-MB-23 1 human breast carcinoma cells. MMP-9 activitywas detected in cells overexpressing NGAL (N-2 and N-5). Thus, itappeared that elevated NGAL expression resulted in an increase in MMP-9activity. Steady state MMP-9 mRNA levels were determined using RT-PCTanalysis and no apparent differences were detected. Expression levels ofendogenous MMP-9 inhibitor, TIMP-1, and a house-keeping gene, GAPGH,were determined and overexpression of NGAL had no apparent influence onmRNA levels of TIMP-1 or GAPDH. Overexpression of NGAL in human breastcarcinoma cells resulted in an increase in MMP-9 activity independent ofchanges in MMP-9 gene transcription.

Discussion

Identification of hMW enzyme complexes in the urine of cancer patients,e.g., enzyme complexes comprising MMP-9 and NGAL, is predictive of TRACand is supported by the following findings: (a) the 125 kDa MMP activityin urine -migrates at the same position as human neutrophil MMP-9/NGALdoes; (b) anti-NGAL antibody successfully detected a 125 kDa proteinband in most of the concentrated urine samples that contain the 125 kDaMMP activity; (c) the same antibody was able to specificallyimmunoprecipitate the 125 kDa MMP activity in urine in aconcentration-dependent manner, without affecting any other MMPactivities. Such evidence agrees with the findings described in U.S.Ser. No. 09/469,637, which is incorporated herein by reference in itsentirety, that the detection of hMW MMPs, as well as MMP-9 and MMP-2,serves as independent predictors of metastatic or organ-confinedcancers, respectively.

NGAL was first identified as a 25 kDa protein that was co-purified withhuman neutrophil gelatinase (Kjeldsen, L., et al. (1993) J Biol Chem.268: 10425-32). Binding of NGAL and MMP-9 results in a gelatinaseactivity of 135 kDa detected in specific granules of human neutrophilstimulated with phorbol myristate acetate (PMA) (Kjeldsen, L. et al.(1993)). NGAL and MMP-9 are stored in specific granules, while MMP-9 isalso present independently in gelatinase granules (Morel, F., et al.,(1994) Biochim Biophys Acta. 1201: 373-80; Kjeldsen, L., et al. (1994)Blood. 83: 799-807; and Borregaard, N. and Cowland, J. B. (1997) Blood.89: 3503-21). However, the MMP-9/NGLA complex detected in urine ofcancer patients are not derived from leukocytes since we havespecifically excluded the urine samples that contain leukocytes.

Interestingly, human NGAL contains sequence similarities to mouse 24p3and rat neu/HER2/c-erbB-2 related lipocalin (NRL), both overexpressed inoncogene mediated cell transformation (Cowland, J. B. and Borregaard, N.(1997) Genomics. 45: 17-23; Hraba-Renevey, S., et al. (1989) Oncogene.4: 601-8; Stoesz, S. P. and Gould, M. N. (1995) Oncogene. 11: 2233-41).Under normal conditions, expression of human NGAL is restricted tobreast, lung, trachea, and bone marrow (Cowland, J. B. and Borregaard,N. (1997) Genomics. 45: 17-23; Stoesz, S. P., et al. (1998) Int. J.Cancer. 79: 565-72). However, elevated levels of NGAL expression hasbeen observed in human breast tumors as well as in adenocarcinomas oflung, colon and pancreas (Stoesz (1998); Friedl, A., et al.(1999)Histochem J. 31: 433-41). An increased production of NGAL can be closelyassociated with cancer disease status, which subsequently contribute tothe elevated levels of MMP-9/NGAL complex in urine. This complex can bedetected with substrate gel electrophoresis as well as antibody-basedassays. As described in U.S. Ser. No. 09/469,637, the presence of the125 kDa MMP activity in urine can serve as an independent multivariatepredictor of cancer metastasis, the identification of this activity asMMP-9/NGAL complex will facilitate the development of a non-invasiveprognosis tool to assess disease status of various cancers.

The origin of the 125 kDa MMP-9/NGAL activity in urine of cancerpatients remains unclear. Given that the glomerular filtration limit isonly 45 kDa, it is unlikely that this large protein complex is directlyfiltered from serum into urine. The possibility that MMP-9/NGAL complexforms after each component was separately filtrated into urine wasinvestigated using in vitro reconstitution assay. The resultsdemonstrate the feasibility of MMP-9/NGAL complex formation ingelatinase buffers with different pH values, as well as, in normalurine. Therefore, it is likely that MMP-9 and NGAL are separatelyexecuted into urine where they form the 125 kDa MMP-9/NGAL complex.

The existence of MMP-9 and NGAL complex in urine was supported by arecent independent study (Monier, F., Clin Chim Acta. 299: 11-23, 2000).Under reducing conditions, MMP-9 and NGAL were separately detected in acontinuous-elution electrophoresis fraction that contains a 115 kDagelatinase activity. The detection of MMP-9 and NGAL in the samefraction shows the observed 115 kDa gelatinase activity as a complex ofMMP-9 and NGAL.

Recent studies have also confirmed that NGAL appears to exert aprotective effect on MMP-9 and prevents MMP-9 from degradation both invitro and in cells. Examples 5 and 6 suggest that the MMP-9-NGAL complexlikely plays an active role in tumor progression.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-80. (canceled)
 81. A method for determining if an individual has atissue remodeling associated condition comprising (a) determining alevel of NGAL present in urine obtained from an individual; and (b)comparing the level of NGAL from the individual's urine with a controllevel of NGAL, wherein a level of NGAL from the individual's urine thatis higher than the control level of NGAL indicates that the individualhas a tissue remodeling associated condition.
 82. A method fordetermining if an individual has a cancer comprising (a) determining alevel of NGAL present in urine obtained from an individual; and (b)comparing the level of NGAL from the individual's urine with a controllevel of NGAL, wherein a level of NGAL from the individual's urine thatis higher than the control level of NGAL indicates that the individualhas a cancer.
 83. The method of claim 81 wherein the tissue remodelingassociated condition is selected from the group consisting of cancers ofthe nervous system, prostate, breast, ovary, retina, lung, skin, kidney,liver, pancreas, uterus, vagina, genito-urinary tract, andgastrointestinal tract.
 84. The method of claim 83 wherein the cancercomprises an epithelial cell cancer.
 85. The method of claim 83 whereinthe cancer comprises a cancer of mesodermal or endodermal origin. 86.The method of claim 85 wherein the cancer comprises bone orhematopoietic cell cancer.
 87. A method for facilitating the diagnosisof an individual for a tissue remodeling-associated condition comprising(a) detecting a lipocalin present in urine obtained from an individual;and (b) correlating the presence or absence of the lipocalin with thepresence or absence of a tissue remodeling-associated condition, therebyfacilitating the diagnosis of the subject for a tissueremodeling-associated condition.
 88. The method of claim 87 wherein thetissue remodeling-associated condition is selected from the groupconsisting of arthritic conditions, obstructive conditions, ulcerativeconditions, and cancers of the nervous system, prostate, breast, ovary,retina, lung, skin, kidney, liver, pancreas, uterus, vagina,genito-urinary tract, and gastrointestinal tract.
 89. The method ofclaim 87 wherein the lipocalin comprises NGAL.
 90. The method of claim87 wherein the cancer comprises an epithelial cell cancer.
 91. Themethod of claim 87 wherein the cancer comprises a cancer of mesodermalor endodermal origin.
 92. The method of claim 91 wherein the cancercomprises bone or hematopoietic cell cancer.